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A subgroup of oropharyngeal squamous cell carcinoma (OPC) is infected with high-risk human papillomavirus (HPV). The object of this study is to evaluate the efficacy of adjuvant chemotherapy with S-1, an oral 5-fluorouracil prodrug, on survival of patients with OPC according to HPV status.Among OPC patients of stage III or IV who received definitive treatment from 1998 to 2008, 38 who were confirmed tumor-free after primary treatment were analyzed. Before 2003, none of the patients received S-1 adjuvant chemotherapy (S-1(-)-group); however, all patients who were eligible were administered S-1 (S-1(+)-group) after 2003. The expression of thymidylate synthase (TYMS) involved in 5-FU metabolism was also examined in protein and mRNA levels.Although there was a trend to disease-free and overall survival benefit in HPV-negative patients with S-1, it did not achieve statistical significance (P = .082 and P = .065, respectively). For the HPV-positive patients, the survivals were similar with or without S-1 administration. TYMS-expression in HPV-positive OPC tissues was significantly higher than in HPV-negative ones in both protein and mRNA levels (P = .0489 and P = .0446, respectively).The current study provides a rationale to plan a randomized trial to compare the efficacy of S-1 according to the HPV status in OPCs.